This paper generated a cell-based functional assay for human glucagon receptor antagonist screening. The human glucagon receptor cDNA was cloned into a mammalian expression vector pCDNA3.1. (h Glucagon receptor /pCDNA3.1). This plasmid was co-transfected with a reporter gene construct (3×CRE/3×MRE/SRE-LUC) in HEK293 cells. Stable cell line expressing both human glucagon receptor and the reporter gene was selected for compounds screening. The assay condition was optimized. Our results demonstrated that this cell-based assay can be optimized for high throughput screening and applied to identify antagonist for glucagon receptor in Chinese Herbs.